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Abstract

Introduction

Glucosamine is an amino-monosaccharide and precursor of glycosaminoglycans, major
components of joint cartilage. Glucosamine has been clinically introduced for the
treatment of osteoarthritis but the data about its protective role in disease are
insufficient. The goal of this study was to investigate the effect of long term administration
of glucosamine on bone resorption and remodeling.

Results

The administration of glucosamine hydrochloride in CIOA mice inhibited loss of glycosaminoglycans
(GAGs) and proteoglycans (PGs) in cartilage, bone erosion and osteophyte formation.
It decreased the levels of soluble RANKL and IL-6 and induced IL-10 increase in the
CIOA joint fluids. Glucosamine limited the number of CD11b positive Ly6G neutrophils
and RANKL positive CD3 T cells in the joint extracts. It suppressed bone resorption
via down-regulation of RANKL expression and affected bone remodeling in CIOA by decreasing
BMP-2, TGF-β3 and pSMAD-2 expression and up-regulating DKK-1 joint levels.

Conclusions

Our data suggest that glucosamine hydrochloride inhibits bone resorption through down-regulation
of RANKL expression in the joints, via reduction of the number of RANKL positive CD3
T cells and the level of sRANKL in the joints extracts. These effects of glucosamine
appear to be critical for the progression of CIOA and result in limited bone remodeling
of the joints.

Introduction

Glucosamine is one of the most abundant amino-monosaccharides immediately phosphorylated
and included in a hexosamine biosynthesis pathway. The end-product of this pathway
is UDP-N-acetylglucosamine, which is important for the synthesis of glycoaminoglycans
and glycolipids. Exogenous glucosamine is bound with high affinity to glucose transporter
GLUT-2 [1] and can induce insulin resistance in adipocytes [2] and skeletal muscle cells [3]. In joint and cartilage, glucosamine can regulate the metabolism of glycosaminoglycans
favoring catabolic processes. Glucosamine expresses a number of in vitro effects on chondrocytes, including stimulation of proteoglycan synthesis, inhibition
of proteoglycan and collagen degradation, suppression of IL-1 induced activation and
decrease of NF-κB activity [4-8]. Glucosamine has anti-inflammatory action suppressing inducible nitric oxide synthase
(iNOS) expression [9,10], neutrophil functions [11], activation of T-lymphoblasts and dendritic cells [12].

Glucosamine has been used for the treatment of osteoarthritis (OA). It is administered
in different pharmacological forms, including sulfate, N-acetyl-glucosamine, or chlorohydrate
salt [13]. Oral application of glucosamine is more frequent, but experimental data about the
effect of its intravenous injection have also been performed [14]. Glucosamine is absorbed from the gastrointestinal tract [15]. Depending on the pharmacological form used, its half-life in serum is from 28 h
to 58 h [14]. Müller-Fassbender et al. have established that glucosamine sulfate is as effective as ibuprofen in patients
with knee OA [16]. Long-term oral treatment with this pharmacological form delayed the progression
and improved the symptoms of knee osteoarthritis acting as a disease modifying agent
[17]. In different trials, it has been reported to exert improvement in OA [18], have a moderate effect [19] or show no difference with a placebo [20,21]. This variation in the results determines a need of more systemic investigations
on the mechanisms of glucosamine action.

OA develops as a result of an imbalance between bone resorption and bone remodeling.
Therefore, we have conducted this study to evaluate the effect of glucosamine on these
processes mainly in the joint. Studies on its systemic effects were not in the focus
of our experiments. Glucosamine was administered in an animal model of OA. We determined
the levels of pro- and anti-inflammatory mediators and the phenotype of cells in the
synovial extracts as well as the expression of resorption and remodeling markers in
the joints.

Materials and methods

Mice

Outbred ICR (CD-2) male mice, 10- to 12-weeks-old, weight 20 to 22 g, were purchased
from the Charles River Laboratories (Wilmington, MA, USA). Mice were maintained on
a 12:12 h light:dark cycle and fed standard diet and tap water ad libitum. All experiments were conducted in accordance with the Bulgarian National Guidelines
for the Care and Use of Laboratory Animals (Decree No. 14/19.07.2000) and were approved
by the Animal Care Committee at the Institute of Microbiology, Sofia.

Collagenase-induced osteoarthritis (CIOA) and treatment

For induction of OA, ICR male mice were injected at right and left knee intra-articular
(i.a.) space with 1 U/10 μl or with 2 U/10 μl of collagenase from Clostridium histolyticum (Sigma-Aldrich, Diesenhofen, Germany) at days 0 and 2. The incidence of OA was approximately
90%. A study to compare the development of CIOA in male and female animals was not
conducted. Control group of animals received i.a. injection of 10 μl endotoxin-free
phosphate-buffered saline (PBS; Lonza, Verviers, Belgium).

D(+)-glucosamine hydrochloride (Glu) and D-glucosamine 2-sulfate sodium salt (GS)
purchased from Sigma-Aldrich (Munich, Germany) were dissolved in sterile PBS and were
administered orally by gavage at a dose of 20 mg/kg/daily. Two groups were treated
with Glu (CIOA + Glu1) or GS (CIOA + GS) for 20 days, starting from Day 7 after the
second collagenase injection and a group of mice with arthritis were fed with PBS
(CIOA). Control groups of mice were i.a. injected with PBS and were untreated (healthy)
or treated with glucosamine hydrochloride (healthy + Glu). One group of mice was treated
with Glu for 20 days starting with the second collagenase injection (CIOA + Glu2).
In another experimental setting CIOA was induced after injection of a higher dose
of collagenase (2 U/mouse at Day 0 and at Day 2). The oral administration of glucosamine
hydrochloride started 7 days after the second collagenase injection and lasted 20
days.

Synovial extracts

Patellae with surrounding soft tissue (tendon and synovium) were excised, and incubated
in 200 μl of serum-free RPMI 1640 medium (Biowhittaker™, Lonza, Verviers, Belgium)
for 2 h at 37°C as described by van de Loo et al. [22]. The washouts were collected separately for each animal and centrifuged at 1,200
× g for 10 minutes. Supernatants were stored at -70°C and used for cytokine assays. Synovial
cells were counted and used for flow cytometry analyses.

Determination of chemokines and interleukins in synovial extracts

The amounts of MIP-1α, RANTES, soluble RANKL, TNF-α, IL-6, IL-4 and IL-10 in synovial
extracts were determined by ELISA using mouse ELISA kits. The samples were assayed
in triplicates. The concentration of chemokines and cytokines was calculated from
a standard curve of the corresponding recombinant mouse protein, using Gen5 Data Analysis
Software (BioTek Instruments, Bad Frienarichsall, Germany) and was presented in pg/ml
supernatant. ELISA kits were with detection limits 8 pg/ml for MIP-1α, 16 pg/ml for
RANTES, 62 pg/ml for RANKL, 32 pg/ml for TNF-α, 20 pg/ml for IL-6, 20 pg/ml for IL-10
(all from PeproTech EC, London, UK) and 10 pg/ml for IL-4 (BD Pharmingen, Erembodegem,
Belgium).

Isolation of peripheral blood mononuclear cells (PBMCs)

Blood was collected by retro-orbital puncture in tubes containing 5 U/ml of heparin.
Blood was mixed with an equal volume of PBS (pH 7.4). After gradient centrifugation
on Histopaque 1083™(Sigma-Aldrich) at 1,400 × g for 40 minutes at room temperature, peripheral blood mononuclear cells (PBMCs) were
carefully collected, washed with PBS, counted and used for flow cytometry analyses.

Histological analyses

Dissected ankle joints were fixed in 10% paraformaldehyde/PBS, decalcified in 5% nitric
acid for one week, dehydrated and embedded in paraffin. Sections (6 μm thickness,
rotary microtome Accu-Cut® SRM™ Sacura Finetek, Tokyo, Japan) were stained with hematoxylin and eosin (H&E),
toluidine blue or safranin O/fast green. Images were captured with a coupled device
camera and exported to Adobe Photoshop 7.0 (Adobe Systems, Munich, Germany). The joint
damage was scored using the semi-quantitative grading and staging system [23]. The severity of damage was graded from 0 (normal joint architecture) to 6 (deformation,
joint margin osteophytes formation and bone remodeling). The extent of the damage
reflecting OA stage was scored from 0 (whole cartilage surface intact) to 4 (more
than 50% of cartilage surface affected). The histological score of joint damage was
obtained after multiplying the grade and stage scores (maximum score 24). Captured
images of the joints were examined for the presence of osteophytes. The osteophyte
area was measured using imaging system software (ImageJ 1.42; Research Services Branch,
NIH, Bethesda, Maryland, USA) and an average value of five joint sections per group
was calculated.

Immunohistochemistry

The sections (6 μm) were permeabilized with 0.1% Triton X-100 in PBS for 20 minutes,
washed with PBS and blocked with 5% bovine serum albumin/PBS for 1 h at room temperature.
The endogenous peroxidase was blocked by 0.3% H2O2 in 60% methanol for 10 minutes. After washing, the sections were incubated for 40
minutes at room temperature with antibodes against RANKL (50 μg/ml; PeproTech EC),
BMP2 (0.1 μg/ml), TGF-β3 (10 μg/ml), pSMAD-2 (20 μg/ml) and DKK-1 (10 μg/ml; all from
Abcam, Cambridge, UK). Isotype antibodies (anti-mouse IgG or anti-rabbit IgG; Sigma-Aldrich)
were used as a background staining control. After washing, the joint sections were
incubated for 10 minutes with biotinylated anti-mouse or anti-rabbit IgGs (Abcam).
Then streptavidin-peroxidase (1:100 diluted; Abcam) was added for 10 minutes. The
sections were washed and incubated with DAB solution kit (3',3'diaminobenzididne kit,
Abcam) for 10 minutes and counterstained with Gill's hematoxylin for 3 minutes. The
number of cells stained positive for the examined proteins was determined by imaging
system software (ImageJ 1.42; Research Services Branch, NIH, USA).

Statistical analyses

Statistical analyses were performed using InStat3.0 and GraphicPad Prism 5.0 (GraphPad
Software Inc., La Jolla, CA, USA). Data are expressed as mean ± standard deviation
(SD). The histological score data and the immunohistochemistry data were analyzed
using the Mann-Whitney U-test. For other data, the differences in mean values between
groups were analyzed by two-tailed Student's t-test. Differences were considered significant when P < 0.05.

Results

Cytokine levels in the synovial extracts at Day 7 of CIOA

At Day 7 of CIOA, we found high levels of pro-inflammatory mediators MIP-1α, RANTES,
soluble RANKL, TNF-α and IL-6 in the synovial extracts. The levels of anti-inflammatory
cytokines IL-4 and IL-10 were nearly undetectable similarly to healthy mice (Table
1).

Table 1. Chemokine and cytokine levels in the joint extracts on Day 7 of CIOA

Effect of glucosamine on the development of CIOA

The severity and progression of CIOA was evaluated by scoring the histological changes
in the joints (grade) and the extent of cartilage involvement in these changes (stage
of CIOA). The repeated injection of 1 U/mouse of collagenase resulted in cartilage
erosion and matrix loss. Histological analyses of H&E stained sections of CIOA joints
showed matrix cracks and fissures extended in the deeper zone of cartilage that contributed
to excavation and cartilage fractures. Reduced density of Toluidine blue staining
in CIOA mice demonstrated marked loss of glycosaminoglycans (GAGs) in the cartilage
(Figure 1b). Safranin O staining showed significant proteoglycan depletion in CIOA joints (Figure
1c). In several CIOA joints we were able to detect osteophyte areas indicative of bone
repair processes, initiated as a result of severe cartilage and matrix loss (Figure
1d). All together our data showed the development of moderate OA, according to the total
histological score of 11.4 ± 4.5 (n = 15, Figure 1e).

Figure 1.Effect of glucosamine on the development of CIOA. CIOA was induced by injection of 1 U/mouse collagenase at Day 0 and Day 2. After
seven days, CIOA mice were orally treated with PBS (CIOA; n = 15), with glucosamine hydrochloride (20 mg/kg/daily; CIOA + Glu1; n = 15) or glucosamine sulfate (20 mg/kg/daily; GS, n = 10) for 20 days, or with glucosamine hydrochloride (20 mg/kg/daily) started with
the second collagenase injection and lasted 20 days (20 mg/kg/daily; CIOA + Glu2;
n = 10). A control group of non-arthritic mice were fed with PBS (healthy; n = 9). (a) Representative joint sections stained with H&E showed fissures and fractures in cartilage
and bone matrix of CIOA mice at Day 30, attenuated by Glu1 and Glu2, but not by GS;
magnification × 40. Staining with Toluidine blue (b) and Safranin O (c) demonstrated GAGs and PG loss in CIOA that was less pronounced in Glu1-treated CIOA
mice; magnification × 100. Glu1 and Glu2 significantly reduced the osteophyte areas
in CIOA mice, while GS was not effective (d). Data are expressed as the mean ± SD from the evaluation of five joint sections/group;
Student's t-test; **P <0.01 vs CIOA group. (e) Glu1 and Glu2 significantly decreased the total histological score in CIOA mice and
nonsignificantly by GS. Total histological score was calculated using the semi-quantitative
grading and staging system. Data are expressed as the mean ± SD from the evaluation
of 10 joints/group; Student's t-test; ***P <0.001 vs CIOA group.

CIOA mice were treated with glucosamine hydrochloride for 20 days, starting with (Glu2)
or 7 days after the second collagenase injection (Glu1). The administration of the
drug under two different schedules (Glu1 and Glu2) had a beneficial effect on the
development and progression of CIOA as demonstrated by inhibited joint damages (Figure
1a) and reduced total histological score (Figure 1e). Mice treated with Glu1 showed less exerted bone erosion, matrix fissures, GAG and
PG loss (Figure 1b, c), and osteophyte areas (Figure 1d). The administration of GS under the schedule of Glu1 group for 20 days did not inhibit
cartilage erosions, matrix loss and osteophyte formation (Figure 1a) and failed to improve histological signs of disease (Figure 1e).

In another set of experiments glucosamine hydrochloride was administered after Day
7 of injection with a higher dose of collagenase (2 U/mouse at Day 0 and at Day 2).
The treatment lasted 20 days (Figure S1b in Additional file 1). At Day 30 all CIOA joints showed bone remodeling and repair with extensive osteophyte
and fibrocartilage formation, denudation, deformation of articular surface and changes
in the joint architecture (Figure S1a in Additional file 1). A high histological score of 22.3 ± 5.2 (maximum 24) demonstrated the development
of severe OA. The administration of glucosamine decreased osteophyte and fibrocartilage
areas in CIOA joints, although this effect did not reach statistical significance
and was observed for some but not for all joint sections (Figure S1b in Additional
file 1).

Additional file 1.Figure S1. Administration of glucosamine in severe CIOA. ICR mice were injected with high dose of collagenase (two i.a. injections with 2
U/mouse at Day 0 and Day 2). After 7 days glucosamine hydrochloride was administered
orally at a dose of 20 mg/kg for 20 days. (a) Representative joint sections stained with H&E showed a mild effect of glucosamine
on osteophyte formation and bone remodeling at Day 30 of severe CIOA. (b) Histological score of CIOA joints was not significantly affected by glucosamine. The
data are expressed as the mean ± SD from two independent experiments involving five
mice per group.

Glucosamine altered the chemokine and interleukin secretion in the synovial extract
of CIOA mice

Several cytokines can affect the severity and progression of OA. We evaluated the
level of MIP-1α, RANTES, TNF-α, soluble RANKL, IL-10 and IL-4 in the synovium extracts
on Day 30 of CIOA (Figure 2). In healthy mice the presence of all mediators was in negligible amounts similar
to the group of non-arthritic mice, treated with Glu1 except for IL-10, whose level
was elevated. Increased levels of MIP-1α, RANTES, TNF-α, soluble RANKL and IL-10 were
established in CIOA mice. Glucosamine markedly enhanced IL-10 in the joint extracts,
while MIP-1α, RANTES and TNF-α were not affected. The administration of the drug for
seven days (or Day 14 of CIOA, insert in Figure 2) significantly decreased soluble RANKL. Low levels of sRANKL were also detected after
long term treatment (20 days) of CIOA mice with glucosamine (Figure 2). In ongoing OA (insert Figure 2), the drug did not affect the level of IL-6, but was able to reduce it after prolonged
administration (20 days; Figure 2).

Glucosamine decreased the number of Ly6G neutrophils and CD3 T cells in the synovial
extract

We performed flow cytometry analyses of synovial cells for the surface expression
of CD3, CD69, Ly6G and CD11b. At Day 30 of CIOA around 30% of synovial cells expressed
neutrophil marker Ly6G, significantly reduced in Glu1 and GS-treated groups (Figure
3a). Low frequencies of single CD11b cells were found in GS and Glu1-treated groups
(Figure 3b). CD3 expressed on 19% of synovial CIOA cells was greatly reduced by Glu1 and more
slightly by GS (Figure 3c). Similar tendency was observed in regard to double positive CD3+/CD69+ cells. The
number of activated cells (3%) was decreased in the synovial extracts of Glu1- and
GS-treated groups (Figure 3d).

Glucosamine changed the expression of TNF-αR and IFN-γR on synovial CD3 T cells

Synovial extracts from CIOA mice contained CD3 positive T cells that can respond to
pro-inflammatory cytokines like TNF-α and IFN-γ after engagement of the particular
receptor. Thus, we next evaluated the expression of TNF-αR and IFN-γR on CD3 T cells.
In order to perform correct flow cytometry analyses, the expression of both receptors
was evaluated within gated CD3 positive cell population in all groups (Figure 4). In healthy mice synovial CD3 T cells expressed TNF-αR1 (mean expression of 1,006
± 345), while CIOA mice showed low surface expression of the receptor (mean expression
of 95 ± 12). In glucosamine-treated mice with CIOA TNF-αR1 expression was comparable
to that in healthy mice (mean expression of 915 ± 102) (Figure 4a). Opposite to these findings, we observed low expression of IFN-γR1 on CD3 T cells
in healthy mice. The surface expression of IFN-γR1 increased in CIOA mice (mean expression
of 1,496 ± 213) which was down-regulated by glucosamine (mean expression of 595 ±
87) (Figure 4b).

Effect of glucosamine on the expression of markers for bone erosion and remodeling

The progression of OA is often due to the lack of balance between bone resorption
and formation. We next evaluated the expression of RANKL, a molecule characteristic
for bone resorption and of BMP2, TGF-α3, pSMAD-2 and DKK-1, molecules indicative for
bone repair and remodeling. The expression of these markers in the joints is shown
in Figure 6 and Table 2. We found single RANKL positive cells in healthy mice. High RANKL expression was
determined at Day 30 of CIOA and in all joint sections with extensive bone erosion.
Glucosamine reduced significantly RANKL positive cells in CIOA joints (Figure 6a, Table 2). The direct action of glucosamine on osteoclasts was determined in vitro. Bone marrow cells were differentiated with M-SCF and RANKL in the presence of increasing
concentrations of the drug. Glucosamine inhibited in a dose-dependent manner osteoclast
differentiation (Figure S2 in Additional file 2). Immunohistochemistry analysis demonstrated BMP-2 staining of osteophyte areas in
CIOA joints that was exerted less in glucosamine-treated group (Figure 6b, Table 2). TGF-α3 positive cells were increased in cartilage of CIOA mice compared to healthy
and glucosamine fed mice (Figure 6c). Downstream signaling of TGF-α3 involved phosphorylation of SMAD-2. The positive
staining for pSMAD-2 found in cartilage of healthy mice was two-fold greater in CIOA
joints. Glucosamine down-regulated the expression of pSMAD-2 (Figure 6d, Table 2). Another protein involved in bone formation is DKK-1, a specific molecule that blocks
Wnt signaling. Expression of DKK-1 was observed in healthy mice. The number of DKK-1
positive cells decreased at Day 30 of CIOA indicating a loss of regulatory signal
to Wnt pathway in favor of bone formation. In the group of glucosamine-treated CIOA
mice we were able to detect DKK-1 positive cells not significantly different from
healthy mice (Figure 6e, Table 2).

Figure 6.Glucosamine altered the expression of bone resorption and bone remodeling markers
in the CIOA joints. Joints of healthy mice, CIOA mice treated with PBS (CIOA) and CIOA mice treated
with glucosamine hydrochloride (CIOA + Glu1) were fixed, decalcified, dehydrated and
embedded in paraffin. The sections (n = 10/mice) were stained with antibodies against RANKL, BMP-2, TGF-β3, pSMAD-2 and
DKK-1. The specific staining was detected using a peroxidase-DAB staining kit. The
positive staining for RANKL in the joints (a), BMP-2 in osteophyte areas (b) and TGF-β3 (c), pSMAD-2 (d) and DKK-1 (e) in cartilage of CIOA mice untreated or treated with glucosamine was indicated with
arrows; magnification × 100.

Additional file 2.Figure S2. Effect of glucosamine on osteoclast differentiation in vitro. Bone marrow cells from healthy mice were isolated, resuspended at 1 × 106/ml in MEM medium (Lonza, Verviers, Belgium) containing 10% FCS and 50 ng/ml of macrophage
colony-stimulating factor and cultured for one day. Osteoclasts were generated after
six days of culture with 100 ng/ml RANKL and 50 ng/ml macrophage colony-stimulating
factor, in the absence or the presence of increasing concentrations of glucosamine
(starting from 5 till 100 μg/ml). Tartrate-resistant acid phosphatase (TRAP) on osteoclasts
was determined by TRAP staining kit (Sigma-Diagnostics, Charleston, WV, USA). The
number of TRAP-positive cells was counted and the data are expressed as the mean ±
SD from three independent experiments.

Discussion

Glucosamine is an agent that improves functional activity and slows the progression
of OA especially of the hip and knee. Most of the clinical studies with glucosamine,
designed to treat OA have shown good or moderate symptomatic efficacy [20,24-26]. Contradictory results have been observed regarding the effectiveness of glucosamine
on pain and disability in OA patients vs placebo patients [27,28]. Such discrepancy is due to the use of different formulations, as hydrochloride or
sulfate salts are claimed to have various efficacies. Additionally, the interpretation
of the results is complicated by differences in the trial duration and in the cohort
studied. Chemically and structurally chloride and sulfate are identical and the nature
of the included salt should not influence their biological effect. Glucosamine hydrochloride
has no beneficial effect in regard to relieving OA pain and disability but the lack
of histological data prevents firm conclusions about its effect on other disease symptoms
[29]. Our histological data showed that glucosamine sulfate was less effective than glucosamine
hydrochloride when both were administered under the same conditions in CIOA.

In the present study, the CIOA animal model has been chosen as relevant to the pathology
of OA patients and the oral route as relevant to the clinical application of glucosamine.
We compared two schedules for 20-day treatment with glucosamine hydrochloride, starting
with the injection of collagenase or seven days thereafter (ongoing OA). In ongoing
OA (Day 7) chemokines and cytokines were elevated in the synovium and histological
signs of cartilage damage were already registered. On Day 30 the disease was defined
as moderate OA, characterized by cartilage erosion, matrix loss, GAG and PG depletion,
and formation of osteophytes. The results demonstrated that glucosamine ameliorated
the degree of joint damage. Lower histological score in glucosamine-treated CIOA group
was related to inhibited bone erosion, as shown by reduced GAG and PG loss, and limited
areas of bone outgrowth at the edges of the joints.

However, limited numbers of investigations have demonstrated the precise mechanism
of glucosamine action in OA. In this study the experiments were focused on the events
in the joint without detailed assessment of the systemic action of glucosamine. In
rat non-arthritic models the drug showed anti-inflammatory action by suppressing iNOS
protein expression in the spleen, lung and peritoneal macrophages [10]. Yet, glucosamine might have systemic action, in that it also suppresses nuclear
factor kappa B activity [30]. Particularly, this pathway is blocked in chondrocytes cultured in the presence of
glucosamine, suggesting that it may suppress inflammatory signaling [7]. Cytokines and growth factors play an important role in the pathology of OA [31,32]. They are produced by the synovial cells, chondrocytes and inflammatory cells, and
later, diffuse to the cartilage through the synovial fluid. Does glucosamine act on
the inflammatory mediators in the synovium? On Day 30 we observed in CIOA mice high
synovial levels of the pro-inflammatory cytokine TNF-α and elevated levels of chemokine
RANTES (CCR5) and chemoattractant protein MIP-1α, an indication of persistent local
inflammation. During ongoing OA, glucosamine was not able to reduce the production
of MIP-1α, RANTES and TNF-α. After seven-day treatment we observed that sRANKL level
in the synovium was lowered compared to untreated mice, but the IL-6 level was not
affected. The substance, however, significantly lowered the level of soluble RANKL
and IL-6, and increased the level of IL-10 in the joint on Day 30. This shows that
various mediators play different roles through the course of the disease. RANKL is
a ligand for RANK expressed on osteoclast precursors. RANKL-RANK interaction triggers
the activation of NF-kB and AP-1 transcription factors that drives osteoclast differentiation
[33]. While IL-10 directly inhibits RANKL-induced osteoclastogenesis [34], IL-6 plays a more complex and dual role in this process. IL-6 can suppress bone
resorption by inhibiting the differentiation of osteoclast progenitors [35] and by down-regulating RANK expression on mature osteoclasts [36]. When IL-6 is at a high level, it binds to soluble IL-6R and can directly induce
RANKL expression on osteoblasts [37] and on synovial fibroblasts that favor bone resorption [38]. In glucosamine-treated mice the reduction of IL-6 and the increase of IL-10 levels
can inhibit the expression of RANKL in the joints. We found only a few RANKL-positive
chondrocytes and synoviocytes in cartilage of glucosamine-treated CIOA mice. In vitro experiments showed that glucosamine dose-dependently inhibited osteoclast differentiation
of bone marrow cells. The membrane-bound RANKL is expressed as a trimeric transmembrane
protein [39], as a truncated ectodomain cleaved by TNF-α convertase and matrix metalloproteinase
14 [40,41] and as a primary secreted form, produced by activated T cells [42]. Our data showed that glucosamine inhibited the expression of RANKL in the joints
and also reduced the level of sRANKL in synovial extract of CIOA mice. Soluble RANKL
directly participate in bone erosion through its excess production by activated CD3
and CD4 cells in synovial fluid in RA patients [43]. Thus, we can hypothesize that diminished levels of sRANKL can be responsible for
suppressed bone erosion in glucosamine-treated CIOA mice. If OA develops as a result
of an imbalance between bone resorption and bone remodeling, the question arises whether
glucosamine can influence another remodeling marker besides RANKL.

In established CIOA, we found intensive formation of osteophytes indicating a prevalence
of bone remodeling. It is reported that cartilage damage is highly associated with
the presence of osteophytes [44], which can be the source of pain and disability in OA [45]. It is not exactly known whether they are formed as a way to compensate for the instability
of destructed bones or as a result of extensive local production of growth factors
[46]. We observed that glucosamine inhibited the formation of osteophytes in CIOA joints.
Simultaneously, we found fewer osteophyte areas positive for BMP-2 in the glucosamine-treated
group. BMP-2, a member of the TGF-β family, can induce osteophyte formation [47] and is expressed at a late stage of endochondral ossification [48,49]. The reduced BMP-2 expression in glucosamine-treated CIOA mice showed that bone remodeling
is initiated but does not progress. Thus, we evaluated the expression of another factor
that can induce osteophyte formation, TGF-β3, which appears earlier than BMP2 in osteophytes
[50]. Increased numbers of cartilage cells positive for TGF-β3 were found at Day 30 of
CIOA. TGF-β3 signaling was active since phosphorylation of downstream molecule SMAD-2
was also detected in the CIOA joints. Davidson et al. have shown that TGF-β3 and pSMAD-2 expression in cartilage decreased with the progression
of a collagenase-induced model of OA and is absent at late stages of severe OA [50]. Such different TGF-β3 and pSMAD-2 expression were observed in moderate and severe
OA induced with different doses of collagenase. The injection of 1 U/mouse resulted
in OA with signs of bone erosion and expression of TGF-β3 on cartilage cells. When
severe OA is induced by repeated injection of 2 U/mouse (Figure S1 in Additional file
1) we were not able to detect TGF-β3 and pSMAD-2 positive cells in the joints, confirming
the observations of Davidson et al. [50]. Down-regulated TGF-β3 and pSMAD-2 expression on cartilage cells may contribute to
inhibited chondrogenesis, altered chondrocyte terminal differentiation and/or reduced
hypertrophy of chondrocytes. In severe OA, the administration of glucosamine was neither
able to affect TGF-β3 signaling in cartilage nor to inhibit bone remodeling and osteophyte
formation. The data indicated that factors other than TGF-β3 and pSMAD-2 can become
important in bone remodeling and repair at Day 30 of CIOA, probably BMP-2.

Wnt signaling is a key factor involved in bone formation. Wnt proteins enhance osteoblast
differentiation and inhibit osteoclast formation [51-53]. DKK-1 is a negative regulator of Wnt signaling [54]. Heterozygous DKK-1(+/-) mice had increased number of osteoblasts and high rates
of bone turnover [55]. In CIOA mice low expression of DKK-1 in the joint indicated increased activation
of Wnt signaling in favor of bone formation. DKK-1 staining was more pronounced in
glucosamine-treated CIOA mice suggesting an increase of bone resorption. Pro-inflammatory
mediators like TNF-α can enhance DKK-1 expression [56]. However, glucosamine was not able to change the level of TNF-α in synovial fluid
of CIOA mice. Probably, a complex interplay between osteoclasts and osteoblasts can
explain this finding. DKK-1 can regulate osteoclastogenesis by enhancing RANKL/RANK
interactions and by down-regulating osteoprotegerin secretion [53,57]. Thus, increased DKK-1 in the glucosamine-treated group can be a mechanism that prevents
excessive bone formation in CIOA mice.

The perpetuation of the inflammatory response in CIOA is related to elevated numbers
of cells in synovial extracts and particularly to influx of neutrophils. Hua et al. showed that glucosamine had a direct effect on neutrophil function. It inhibited
superoxide generation and phagocytosis of neutrophils, and also can suppress formyl-Met-Leu-Phe-induced
up-regulation of CD11b on these cells [11]. When coupled to FcγRs, CD11b can trigger p38 mitogen-activated protein kinase pathways,
important for actin polymerization and chemotaxis of neutrophils [58]. A recent study has shown that neutrophils expressed RANKL [59] and can activate directly osteoclasts in a coculture system [60]. Glucosamine via inhibition of neutrophil chemotaxis to synovium can have an impact
on osteoclastogenesis and bone destruction. CD11b is also expressed on monocytes,
cells that are the source of osteoclast precursors. An inhibited number of CD11b positive
cells in the synovial extracts from glucosamine-treated mice can result in reduced
numbers of osteoclast precursors and in turn, in restricted osteoclastogenesis. The
role of T cells in the pathology of OA should not be neglected as T cell infiltrates
are frequently detected in the synovial membrane of patients with OA [61]. In CIOA mice we found an increased percentage of cells expressing CD3 (18%). However,
only 3% of them were activated and expressed CD69. CD3 T cells showed low TNF-αR expression,
even lesser than that on CD3 cells from healthy mice, while they were highly positive
for RANKL. Recently, it has been shown that TNF-αR plays an important role in RANKL
signaling, since it can compete with RANKL for the intracellular molecules TNF receptor
associated factors 2, 5, and 6 [62]. When more TNF-αRs are engaged, RANKL signaling is less sensitive and expression
of RANKL decreased. In support to this finding, we have observed up-regulation of
TNF-αR but inhibited RANKL expression on synovial CD3 T cells from glucosamine-treated
CIOA mice. RANKL-positive CD3 T cells in synovium can interact with osteoclasts, dendritic
cells and/or neutrophils, all expressing RANK and thus, promoting local pro-inflammatory
response and bone resorption. While glucosamine treatment reduced the number of synovial
RANKL positive cells, such a reduction was not observed in the periphery. Probably,
fewer RANKL-positive CD3 T cells infiltrate CIOA joints and thus, more of them were
found in the periphery. Recently, it has been demonstrated that the recruitment of
IFN-γR1 into the immunologic synapses of helper T (Th) cells correlates with their
capacity to differentiate into Th1 effector cells [63]. In our study, glucosamine inhibited the expression of IFN-γR1 on CD3 T cells suggesting
that the drug can have an impact on Th1 cell differentiation and on the perpetuation
of inflammatory processes in OA. Histologically, in the OA synovium a mixed inflammatory
infiltrate consisting mainly of macrophages is observed [64]. OA synovial macrophages exhibit an activated phenotype and they mediate osteophyte
formation and other OA-related pathology [65]. It is an important question: Which cell type in the OA synovium is predominantly
affected by glucosamine? In order to answer the question further experiments should
be conducted, possibly by depletion of neutrophils, macrophages or CD3 cells.

Conclusions

Our data show that glucosamine acted on the arthritic process in joints through inhibition
of neutrophils and, at least, partially of T cells, particularly of CD3. The substance
attenuates bone resorption in moderate OA via inhibition of RANKL expression in the
joint, reduction of sRANKL and IL-6 levels, and increase of IL-10 amount in the synovial
fluid of CIOA mice. The drug diminishes the number of RANKL positive CD3 T cells in
the synovial extract and changes their RANKL expression. Reduced TGF-β3 and pSMAD-2
signaling in glucosamine-treated mice are in favor of inhibited bone remodeling and
formation of BMP-2 positive osteophytes. The substance is able to limit the excessive
bone formation at the late stage of disease by increased expression of DKK-1 in the
CIOA joints. The data show that glucosamine ameliorates CIOA progression by regulating
the degree of bone resorption and bone remodeling.